Ethanol potentiates the function of serotonin type 3 (5-HT3) receptors in various types of neurons and several studies have suggested that 5-HT3 receptors may be involved in alcohol preference and reward mechanisms. However, the molecular basis for alcohol modulation of 5-HT3 receptor function has not been determined. Previously, using a chimeric nicotinic-serotonergic receptor, we found evidence that alcohol effects on receptor function involve the extracellular N-terminal domain of the receptor (Mol Pharmacol 1996;50:1010-1016). We are now studying the effect of single amino acid mutations in the N-terminal domain of the 5-HT3 receptor on the function and ethanol sensitivity of the receptor. In most experiments, recombinant wild-type (WT) or mutant 5-HT3 receptors were expressed in Xenopus oocytes and their function was studied using two-electrode voltage-clamp. Point-mutations of the arginine at amino acid 222 (R222) in the N-terminal domain (adjacent to the first transmembrane domain) of the 5-HT3 receptor, altered the EC50 value of the 5-HT concentration-response curve. Some point-mutations at amino acid 222 resulted in spontaneous opening of the 5-HT3 receptor-channel. For these mutant receptors, ethanol activated an inward current in the absence of agonist. In addition, the amplitude of current activated by ethanol in the absence of agonist correlates with the amplitude of current resulting from spontaneous channel openings, suggesting that these mutations induce ethanol sensitivity of the receptor by the induction of spontaneous channel openings. Other point-mutations enhanced ethanol potentiation of agonist-activated responses; for these mutations, ethanol did not activate significant inward current in the absence of agonist. For these receptors, the magnitude of the ethanol potentiation of agonist-activated responses correlates inversely with the EC50 values of the 5-HT concentration-response curves, suggesting that these mutations modulate ethanol sensitivity of the receptor by altering the apparent agonist affinity of the receptor. Kinetic analysis of one of these receptors, R222A, expressed in HEK 293 cells (using whole-cell patch-clamp recording in combination with rapid extracellular solution exchange) revealed that the mutation greatly increased the rate of channel opening (activation) but did not affect the rate of channel closing (deactivation). The observations suggest that amino acid 222 is involved in gating the 5-HT3 receptor-channel and thereby allosterically affects ethanol sensitivity of the receptor. Experiments are also in progress to elucidate the molecular determinants for alcohol and neuroactive substance sensitivity of other neurotransmitter-gated membrane ion channels, such as N-methyl-D-aspartate (NMDA) receptors, gamma-aminobutyric type A (GABA-A) receptors, nicotinic acetylcholine (nACh) receptors, glycine receptors and ATP receptors. These studies hold the promise that such molecular biological approaches will advance our knowledge of the molecular basis of alcohol and neuroactive substance action in the nervous system and provide a foundation for understanding the molecular basis of alcohol abuse and alcoholism.